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HEADER BIOSYNTHETIC PROTEIN 07-AUG-23 8TQG
TITLE CRYSTAL STRUCTURE OF MTB PKS13 THIOESTERASE DOMAIN IN COMPLEX WITH
TITLE 2 INHIBITOR X20419
COMPND MOL_ID: 1;
COMPND 2 MOLECULE: POLYKETIDE SYNTHASE PKS13;
COMPND 3 CHAIN: A;
COMPND 4 SYNONYM: TERMINATION POLYKETIDE SYNTHASE;
COMPND 5 EC: 2.3.1.-;
COMPND 6 ENGINEERED: YES
SOURCE MOL_ID: 1;
SOURCE 2 ORGANISM_SCIENTIFIC: MYCOBACTERIUM TUBERCULOSIS;
SOURCE 3 ORGANISM_TAXID: 1773;
SOURCE 4 GENE: PKS13, RV3800C;
SOURCE 5 EXPRESSION_SYSTEM: ESCHERICHIA COLI;
SOURCE 6 EXPRESSION_SYSTEM_TAXID: 562
KEYWDS POLYKETIDE SYNTHASE, THIOESTERASE, BIOSYNTHETIC PROTEIN
EXPDTA X-RAY DIFFRACTION
AUTHOR I.V.KRIEGER,J.C.SACCHETTINI
REVDAT 1 24-APR-24 8TQG 0
JRNL AUTH I.V.KRIEGER,S.YALAMANCHILI,P.DICKSON,C.A.ENGELHART,
JRNL AUTH 2 M.D.ZIMMERMAN,J.WOOD,E.CLARY,J.NGUYEN,N.THORNTON,
JRNL AUTH 3 P.A.CENTRELLA,B.CHAN,J.W.CUOZZO,M.GENGENBACHER,M.A.GUIE,
JRNL AUTH 4 J.P.GUILINGER,C.BIENSTOCK,H.HARTL,C.D.HUPP,R.JETSON,T.SATOH,
JRNL AUTH 5 J.T.S.YEOMAN,Y.ZHANG,V.DARTOIS,D.SCHNAPPINGER,A.D.KEEFE,
JRNL AUTH 6 J.C.SACCHETTINI
JRNL TITL INHIBITORS OF THE THIOESTERASE ACTIVITY OF MYCOBACTERIUM
JRNL TITL 2 TUBERCULOSIS PKS13 DISCOVERED USING DNA-ENCODED CHEMICAL
JRNL TITL 3 LIBRARY SCREENING.
JRNL REF ACS INFECT DIS. 2024
JRNL REFN ESSN 2373-8227
JRNL PMID 38577994
JRNL DOI 10.1021/ACSINFECDIS.3C00592
REMARK 2
REMARK 2 RESOLUTION. 2.20 ANGSTROMS.
REMARK 3
REMARK 3 REFINEMENT.
REMARK 3 PROGRAM : PHENIX 1.20.1_4487
REMARK 3 AUTHORS : PAUL ADAMS,PAVEL AFONINE,VINCENT CHEN,IAN
REMARK 3 : DAVIS,KRESHNA GOPAL,RALF GROSSE-KUNSTLEVE,
REMARK 3 : LI-WEI HUNG,ROBERT IMMORMINO,TOM IOERGER,
REMARK 3 : AIRLIE MCCOY,ERIK MCKEE,NIGEL MORIARTY,
REMARK 3 : REETAL PAI,RANDY READ,JANE RICHARDSON,
REMARK 3 : DAVID RICHARDSON,TOD ROMO,JIM SACCHETTINI,
REMARK 3 : NICHOLAS SAUTER,JACOB SMITH,LAURENT
REMARK 3 : STORONI,TOM TERWILLIGER,PETER ZWART
REMARK 3
REMARK 3 REFINEMENT TARGET : GEOSTD + MONOMER LIBRARY + CDL V1.2
REMARK 3
REMARK 3 DATA USED IN REFINEMENT.
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 2.20
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 59.29
REMARK 3 MIN(FOBS/SIGMA_FOBS) : 1.340
REMARK 3 COMPLETENESS FOR RANGE (%) : 98.4
REMARK 3 NUMBER OF REFLECTIONS : 15293
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT.
REMARK 3 R VALUE (WORKING + TEST SET) : 0.246
REMARK 3 R VALUE (WORKING SET) : 0.242
REMARK 3 FREE R VALUE : 0.313
REMARK 3 FREE R VALUE TEST SET SIZE (%) : 5.200
REMARK 3 FREE R VALUE TEST SET COUNT : 796
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT (IN BINS).
REMARK 3 BIN RESOLUTION RANGE COMPL. NWORK NFREE RWORK RFREE
REMARK 3 1 59.2900 - 4.0000 0.99 2582 161 0.1906 0.2612
REMARK 3 2 4.0000 - 3.1700 0.98 2428 125 0.2454 0.3198
REMARK 3 3 3.1700 - 2.7700 0.99 2388 144 0.2831 0.3878
REMARK 3 4 2.7700 - 2.5200 0.97 2378 114 0.3073 0.3959
REMARK 3 5 2.5200 - 2.3400 0.99 2369 130 0.3360 0.4207
REMARK 3 6 2.3400 - 2.2000 0.98 2352 122 0.3916 0.4437
REMARK 3
REMARK 3 BULK SOLVENT MODELLING.
REMARK 3 METHOD USED : FLAT BULK SOLVENT MODEL
REMARK 3 SOLVENT RADIUS : 1.10
REMARK 3 SHRINKAGE RADIUS : 0.90
REMARK 3 K_SOL : NULL
REMARK 3 B_SOL : NULL
REMARK 3
REMARK 3 ERROR ESTIMATES.
REMARK 3 COORDINATE ERROR (MAXIMUM-LIKELIHOOD BASED) : 0.391
REMARK 3 PHASE ERROR (DEGREES, MAXIMUM-LIKELIHOOD BASED) : 39.364
REMARK 3
REMARK 3 B VALUES.
REMARK 3 FROM WILSON PLOT (A**2) : 49.04
REMARK 3 MEAN B VALUE (OVERALL, A**2) : 62.93
REMARK 3 OVERALL ANISOTROPIC B VALUE.
REMARK 3 B11 (A**2) : NULL
REMARK 3 B22 (A**2) : NULL
REMARK 3 B33 (A**2) : NULL
REMARK 3 B12 (A**2) : NULL
REMARK 3 B13 (A**2) : NULL
REMARK 3 B23 (A**2) : NULL
REMARK 3
REMARK 3 TWINNING INFORMATION.
REMARK 3 FRACTION: NULL
REMARK 3 OPERATOR: NULL
REMARK 3
REMARK 3 DEVIATIONS FROM IDEAL VALUES.
REMARK 3 RMSD COUNT
REMARK 3 BOND : 0.008 2287
REMARK 3 ANGLE : 1.008 3110
REMARK 3 CHIRALITY : 0.060 324
REMARK 3 PLANARITY : 0.012 412
REMARK 3 DIHEDRAL : 9.285 331
REMARK 3
REMARK 3 TLS DETAILS
REMARK 3 NUMBER OF TLS GROUPS : NULL
REMARK 3
REMARK 3 NCS DETAILS
REMARK 3 NUMBER OF NCS GROUPS : NULL
REMARK 3
REMARK 3 OTHER REFINEMENT REMARKS: NULL
REMARK 4
REMARK 4 8TQG COMPLIES WITH FORMAT V. 3.30, 13-JUL-11
REMARK 100
REMARK 100 THIS ENTRY HAS BEEN PROCESSED BY RCSB ON 08-AUG-23.
REMARK 100 THE DEPOSITION ID IS D_1000276391.
REMARK 200
REMARK 200 EXPERIMENTAL DETAILS
REMARK 200 EXPERIMENT TYPE : X-RAY DIFFRACTION
REMARK 200 DATE OF DATA COLLECTION : 28-SEP-21
REMARK 200 TEMPERATURE (KELVIN) : 100
REMARK 200 PH : 8.5
REMARK 200 NUMBER OF CRYSTALS USED : 1
REMARK 200
REMARK 200 SYNCHROTRON (Y/N) : Y
REMARK 200 RADIATION SOURCE : APS
REMARK 200 BEAMLINE : 23-ID-B
REMARK 200 X-RAY GENERATOR MODEL : NULL
REMARK 200 MONOCHROMATIC OR LAUE (M/L) : M
REMARK 200 WAVELENGTH OR RANGE (A) : 1.03317
REMARK 200 MONOCHROMATOR : NULL
REMARK 200 OPTICS : NULL
REMARK 200
REMARK 200 DETECTOR TYPE : PIXEL
REMARK 200 DETECTOR MANUFACTURER : DECTRIS EIGER X 16M
REMARK 200 INTENSITY-INTEGRATION SOFTWARE : HKL-2000
REMARK 200 DATA SCALING SOFTWARE : HKL-2000
REMARK 200
REMARK 200 NUMBER OF UNIQUE REFLECTIONS : 15367
REMARK 200 RESOLUTION RANGE HIGH (A) : 2.200
REMARK 200 RESOLUTION RANGE LOW (A) : 66.630
REMARK 200 REJECTION CRITERIA (SIGMA(I)) : NULL
REMARK 200
REMARK 200 OVERALL.
REMARK 200 COMPLETENESS FOR RANGE (%) : 99.0
REMARK 200 DATA REDUNDANCY : 6.200
REMARK 200 R MERGE (I) : 0.11300
REMARK 200 R SYM (I) : NULL
REMARK 200 FOR THE DATA SET : 8.9000
REMARK 200
REMARK 200 IN THE HIGHEST RESOLUTION SHELL.
REMARK 200 HIGHEST RESOLUTION SHELL, RANGE HIGH (A) : 2.20
REMARK 200 HIGHEST RESOLUTION SHELL, RANGE LOW (A) : 2.27
REMARK 200 COMPLETENESS FOR SHELL (%) : NULL
REMARK 200 DATA REDUNDANCY IN SHELL : NULL
REMARK 200 R MERGE FOR SHELL (I) : 2.24000
REMARK 200 R SYM FOR SHELL (I) : NULL
REMARK 200 FOR SHELL : NULL
REMARK 200
REMARK 200 DIFFRACTION PROTOCOL: SINGLE WAVELENGTH
REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT
REMARK 200 SOFTWARE USED: MOLREP
REMARK 200 STARTING MODEL: NULL
REMARK 200
REMARK 200 REMARK: NULL
REMARK 280
REMARK 280 CRYSTAL
REMARK 280 SOLVENT CONTENT, VS (%): 45.79
REMARK 280 MATTHEWS COEFFICIENT, VM (ANGSTROMS**3/DA): 2.27
REMARK 280
REMARK 280 CRYSTALLIZATION CONDITIONS: 0.1 M TRIS-HCL, 2.0-1.8 M AMMONIUM
REMARK 280 SULFATE, 2%-5% V/V PPG P400, PH 8.5, VAPOR DIFFUSION, HANGING
REMARK 280 DROP, TEMPERATURE 290K
REMARK 290
REMARK 290 CRYSTALLOGRAPHIC SYMMETRY
REMARK 290 SYMMETRY OPERATORS FOR SPACE GROUP: P 41 21 2
REMARK 290
REMARK 290 SYMOP SYMMETRY
REMARK 290 NNNMMM OPERATOR
REMARK 290 1555 X,Y,Z
REMARK 290 2555 -X,-Y,Z+1/2
REMARK 290 3555 -Y+1/2,X+1/2,Z+1/4
REMARK 290 4555 Y+1/2,-X+1/2,Z+3/4
REMARK 290 5555 -X+1/2,Y+1/2,-Z+1/4
REMARK 290 6555 X+1/2,-Y+1/2,-Z+3/4
REMARK 290 7555 Y,X,-Z
REMARK 290 8555 -Y,-X,-Z+1/2
REMARK 290
REMARK 290 WHERE NNN -> OPERATOR NUMBER
REMARK 290 MMM -> TRANSLATION VECTOR
REMARK 290
REMARK 290 CRYSTALLOGRAPHIC SYMMETRY TRANSFORMATIONS
REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THE ATOM/HETATM
REMARK 290 RECORDS IN THIS ENTRY TO PRODUCE CRYSTALLOGRAPHICALLY
REMARK 290 RELATED MOLECULES.
REMARK 290 SMTRY1 1 1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY2 1 0.000000 1.000000 0.000000 0.00000
REMARK 290 SMTRY3 1 0.000000 0.000000 1.000000 0.00000
REMARK 290 SMTRY1 2 -1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY2 2 0.000000 -1.000000 0.000000 0.00000
REMARK 290 SMTRY3 2 0.000000 0.000000 1.000000 64.97650
REMARK 290 SMTRY1 3 0.000000 -1.000000 0.000000 33.31300
REMARK 290 SMTRY2 3 1.000000 0.000000 0.000000 33.31300
REMARK 290 SMTRY3 3 0.000000 0.000000 1.000000 32.48825
REMARK 290 SMTRY1 4 0.000000 1.000000 0.000000 33.31300
REMARK 290 SMTRY2 4 -1.000000 0.000000 0.000000 33.31300
REMARK 290 SMTRY3 4 0.000000 0.000000 1.000000 97.46475
REMARK 290 SMTRY1 5 -1.000000 0.000000 0.000000 33.31300
REMARK 290 SMTRY2 5 0.000000 1.000000 0.000000 33.31300
REMARK 290 SMTRY3 5 0.000000 0.000000 -1.000000 32.48825
REMARK 290 SMTRY1 6 1.000000 0.000000 0.000000 33.31300
REMARK 290 SMTRY2 6 0.000000 -1.000000 0.000000 33.31300
REMARK 290 SMTRY3 6 0.000000 0.000000 -1.000000 97.46475
REMARK 290 SMTRY1 7 0.000000 1.000000 0.000000 0.00000
REMARK 290 SMTRY2 7 1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY3 7 0.000000 0.000000 -1.000000 0.00000
REMARK 290 SMTRY1 8 0.000000 -1.000000 0.000000 0.00000
REMARK 290 SMTRY2 8 -1.000000 0.000000 0.000000 0.00000
REMARK 290 SMTRY3 8 0.000000 0.000000 -1.000000 64.97650
REMARK 290
REMARK 290 REMARK: NULL
REMARK 300
REMARK 300 BIOMOLECULE: 1
REMARK 300 SEE REMARK 350 FOR THE AUTHOR PROVIDED AND/OR PROGRAM
REMARK 300 GENERATED ASSEMBLY INFORMATION FOR THE STRUCTURE IN
REMARK 300 THIS ENTRY. THE REMARK MAY ALSO PROVIDE INFORMATION ON
REMARK 300 BURIED SURFACE AREA.
REMARK 350
REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWN
REMARK 350 BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE
REMARK 350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS
REMARK 350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND
REMARK 350 CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN.
REMARK 350
REMARK 350 BIOMOLECULE: 1
REMARK 350 AUTHOR DETERMINED BIOLOGICAL UNIT: MONOMERIC
REMARK 350 APPLY THE FOLLOWING TO CHAINS: A
REMARK 350 BIOMT1 1 1.000000 0.000000 0.000000 0.00000
REMARK 350 BIOMT2 1 0.000000 1.000000 0.000000 0.00000
REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000
REMARK 465
REMARK 465 MISSING RESIDUES
REMARK 465 THE FOLLOWING RESIDUES WERE NOT LOCATED IN THE
REMARK 465 EXPERIMENT. (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN
REMARK 465 IDENTIFIER; SSSEQ=SEQUENCE NUMBER; I=INSERTION CODE.)
REMARK 465
REMARK 465 M RES C SSSEQI
REMARK 465 SER A 1448
REMARK 465 ASN A 1449
REMARK 465 ALA A 1450
REMARK 465 SER A 1728
REMARK 465 GLU A 1729
REMARK 465 VAL A 1730
REMARK 465 GLY A 1731
REMARK 465 LYS A 1732
REMARK 465 GLN A 1733
REMARK 500
REMARK 500 GEOMETRY AND STEREOCHEMISTRY
REMARK 500 SUBTOPIC: TORSION ANGLES
REMARK 500
REMARK 500 TORSION ANGLES OUTSIDE THE EXPECTED RAMACHANDRAN REGIONS:
REMARK 500 (M=MODEL NUMBER; RES=RESIDUE NAME; C=CHAIN IDENTIFIER;
REMARK 500 SSEQ=SEQUENCE NUMBER; I=INSERTION CODE).
REMARK 500
REMARK 500 STANDARD TABLE:
REMARK 500 FORMAT:(10X,I3,1X,A3,1X,A1,I4,A1,4X,F7.2,3X,F7.2)
REMARK 500
REMARK 500 EXPECTED VALUES: GJ KLEYWEGT AND TA JONES (1996). PHI/PSI-
REMARK 500 CHOLOGY: RAMACHANDRAN REVISITED. STRUCTURE 4, 1395 - 1400
REMARK 500
REMARK 500 M RES CSSEQI PSI PHI
REMARK 500 PRO A1492 155.74 -43.69
REMARK 500 ALA A1493 -83.85 -55.77
REMARK 500 ASP A1494 43.41 -79.21
REMARK 500 SER A1533 -140.36 62.53
REMARK 500 PHE A1590 34.33 -90.65
REMARK 500 ASN A1591 -133.63 49.18
REMARK 500 VAL A1592 70.42 55.59
REMARK 500 PRO A1595 -155.32 -69.76
REMARK 500 ILE A1695 -5.13 -143.54
REMARK 500 ALA A1718 -43.49 173.99
REMARK 500 ASP A1725 6.14 -59.45
REMARK 500
REMARK 500 REMARK: NULL
DBREF 8TQG A 1451 1733 UNP I6X8D2 PKS13_MYCTU 1451 1733
SEQADV 8TQG SER A 1448 UNP I6X8D2 EXPRESSION TAG
SEQADV 8TQG ASN A 1449 UNP I6X8D2 EXPRESSION TAG
SEQADV 8TQG ALA A 1450 UNP I6X8D2 EXPRESSION TAG
SEQRES 1 A 286 SER ASN ALA GLN ILE ASP GLY PHE VAL ARG THR LEU ARG
SEQRES 2 A 286 ALA ARG PRO GLU ALA GLY GLY LYS VAL PRO VAL PHE VAL
SEQRES 3 A 286 PHE HIS PRO ALA GLY GLY SER THR VAL VAL TYR GLU PRO
SEQRES 4 A 286 LEU LEU GLY ARG LEU PRO ALA ASP THR PRO MET TYR GLY
SEQRES 5 A 286 PHE GLU ARG VAL GLU GLY SER ILE GLU GLU ARG ALA GLN
SEQRES 6 A 286 GLN TYR VAL PRO LYS LEU ILE GLU MET GLN GLY ASP GLY
SEQRES 7 A 286 PRO TYR VAL LEU VAL GLY TRP SER LEU GLY GLY VAL LEU
SEQRES 8 A 286 ALA TYR ALA CYS ALA ILE GLY LEU ARG ARG LEU GLY LYS
SEQRES 9 A 286 ASP VAL ARG PHE VAL GLY LEU ILE ASP ALA VAL ARG ALA
SEQRES 10 A 286 GLY GLU GLU ILE PRO GLN THR LYS GLU GLU ILE ARG LYS
SEQRES 11 A 286 ARG TRP ASP ARG TYR ALA ALA PHE ALA GLU LYS THR PHE
SEQRES 12 A 286 ASN VAL THR ILE PRO ALA ILE PRO TYR GLU GLN LEU GLU
SEQRES 13 A 286 GLU LEU ASP ASP GLU GLY GLN VAL ARG PHE VAL LEU ASP
SEQRES 14 A 286 ALA VAL SER GLN SER GLY VAL GLN ILE PRO ALA GLY ILE
SEQRES 15 A 286 ILE GLU HIS GLN ARG THR SER TYR LEU ASP ASN ARG ALA
SEQRES 16 A 286 ILE ASP THR ALA GLN ILE GLN PRO TYR ASP GLY HIS VAL
SEQRES 17 A 286 THR LEU TYR MET ALA ASP ARG TYR HIS ASP ASP ALA ILE
SEQRES 18 A 286 MET PHE GLU PRO ARG TYR ALA VAL ARG GLN PRO ASP GLY
SEQRES 19 A 286 GLY TRP GLY GLU TYR VAL SER ASP LEU GLU VAL VAL PRO
SEQRES 20 A 286 ILE GLY GLY GLU HIS ILE GLN ALA ILE ASP GLU PRO ILE
SEQRES 21 A 286 ILE ALA LYS VAL GLY GLU HIS MET SER ARG ALA LEU GLY
SEQRES 22 A 286 GLN ILE GLU ALA ASP ARG THR SER GLU VAL GLY LYS GLN
HET JR0 A1801 46
HET SO4 A1802 5
HET SO4 A1803 5
HETNAM JR0 N-BENZYL-2-{4-[4-(4,5-DIMETHOXY-1H-INDOLE-2-CARBONYL)
HETNAM 2 JR0 PIPERAZINE-1-CARBONYL]PIPERIDIN-1-YL}-6-
HETNAM 3 JR0 METHYLPYRIMIDINE-4-CARBOXAMIDE
HETNAM SO4 SULFATE ION
FORMUL 2 JR0 C34 H39 N7 O5
FORMUL 3 SO4 2(O4 S 2-)
FORMUL 5 HOH *13(H2 O)
HELIX 1 AA1 SER A 1480 VAL A 1483 5 4
HELIX 2 AA2 TYR A 1484 ARG A 1490 1 7
HELIX 3 AA3 SER A 1506 GLY A 1523 1 18
HELIX 4 AA4 SER A 1533 LEU A 1549 1 17
HELIX 5 AA5 THR A 1571 PHE A 1590 1 20
HELIX 6 AA6 GLU A 1600 LEU A 1605 1 6
HELIX 7 AA7 ASP A 1606 GLN A 1620 1 15
HELIX 8 AA8 PRO A 1626 ALA A 1646 1 21
HELIX 9 AA9 HIS A 1664 GLU A 1671 1 8
HELIX 10 AB1 PRO A 1672 VAL A 1676 5 5
HELIX 11 AB2 ILE A 1707 ASP A 1725 1 19
SHEET 1 AA1 8 ILE A1452 ASP A1453 0
SHEET 2 AA1 8 VAL A1456 ARG A1460 -1 O VAL A1456 N ASP A1453
SHEET 3 AA1 8 MET A1497 PHE A1500 -1 O GLY A1499 N ARG A1457
SHEET 4 AA1 8 VAL A1471 PHE A1474 1 N VAL A1473 O PHE A1500
SHEET 5 AA1 8 TYR A1527 TRP A1532 1 O VAL A1530 N PHE A1472
SHEET 6 AA1 8 VAL A1553 ILE A1559 1 O ILE A1559 N GLY A1531
SHEET 7 AA1 8 VAL A1655 MET A1659 1 O TYR A1658 N LEU A1558
SHEET 8 AA1 8 LEU A1690 PRO A1694 1 O GLU A1691 N LEU A1657
CISPEP 1 GLY A 1525 PRO A 1526 0 -5.41
CISPEP 2 GLU A 1705 PRO A 1706 0 0.56
CRYST1 66.626 66.626 129.953 90.00 90.00 90.00 P 41 21 2 8
ORIGX1 1.000000 0.000000 0.000000 0.00000
ORIGX2 0.000000 1.000000 0.000000 0.00000
ORIGX3 0.000000 0.000000 1.000000 0.00000
SCALE1 0.015009 0.000000 0.000000 0.00000
SCALE2 0.000000 0.015009 0.000000 0.00000
SCALE3 0.000000 0.000000 0.007695 0.00000
TER 2178 THR A1727
MASTER 253 0 3 11 8 0 0 6 2246 1 56 22
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